Stem cells are immature, unspecialized cells that renew themselves for long periods through cell division. Under certain conditions, they can differentiate into mature, functional cells. Human embryonic stem cells (hESC) are derived from early surplus human blastocysts1. Human ES cells are unique stem cells since they can self-renew infinitely in culture, and since they have a remarkable potential to develop into extraembryonic lineages as well as all somatic cells and tissues of the human body1.
Given the unique properties of hESC, they are expected to have far-reaching applications in the areas of basic scientific research, pharmacology, and regenerative medicine. Human ES cell lines can provide a powerful in vitro model for the study of the molecular and cellular biology of early human development, for functional genomics, drug screening, and discovery. They may serve for toxicology and teratogenicity high throughput screening. Since hESC can self-renew indefinitely and can differentiate into any cell type, they can serve as a renewable, unlimited donor source of functionally mature differentiated cells or tissues for transplantation therapy. In addition, transplanted genetically-modified hESC can serve as vectors to carry and express genes in target organs in the course of gene therapy.
While the promise of hESC for basic scientific research pharmacology and regenerative medicine is remarkable, the exploitation of hESC for most applications depends upon further development. Improved control of the growth of undifferentiated hESC, the development of bulk feeder-free cultures of undifferentiated cells, the development of animal-free culture systems, and the development of methods and tools which direct the differentiation and generate pure cultures of mature functional cells of a specific type are required.
At present, a few culture systems are most commonly used to propagate undifferentiated hESC1-3. In the initial culture system that was developed, undifferentiated hESC are cultured in serum-containing medium as colonies, upon a layer of fibroblast feeder cells (of mouse1 or human origin4, 10) It is possible to remove all animal products from this culture system and replace them with those from a human source5. It was found that in this system the cells are propagated as clumps on a small scale, which does not allow efficient cloning2.
An alternative culture system for use in the proliferation of undifferentiated growth of hESC comprises a culture matrix comprising extracellular matrix (ECM) that may be prepared from feeder cells or other sources and a conditioned medium being preconditioned by feeder cells. The suggested leading cells in the feeder cells include primary mouse embryonic fibroblasts (PMEF), a mouse embryonic fibroblast cell line (MEF), murine foetal fibroblasts (MFF), human embryonic fibroblasts (HEF), human foetal muscle (HFM), human foetal skin cells (HFS), human adult skin cells, human foreskin fibroblasts (HFF)9, human adult Fallopian tubal epithelial cells (HAFT), or human marrow stromal cells (HMSC).
Another alternative culture system that was developed and used extensively is a serum-free system that includes the knockout (KO) medium supplemented with knockout serum replacement (KOSR) and FGF2. This system allows cloning of undifferentiated hESC, although at a low efficiency2. Undifferentiated cells are cultured as flat colonies and may be propagated mechanically as small clusters or single cells (by using trypsin6).
Knockout serum replacement (KOSR) (Gibco) is a chemically defined, serum-free culture medium supplement used as a substitute for animal-based serum in KO-DMEM-based culture systems (KO-DMEM e.g. of catalog no. 10829-018 Gibco 1998/1999) for propagating stem cells. KOSR can efficiently promote the growth and maintenance of undifferentiated embryonic stem cells and therefore may replace the supplementation with fetal bovine serum (FBS)(21). KO-DMEM may replace traditional DMEM in either FBS- or KOSR-supplemented cultures(21).
Undifferentiated propagation of adherent colonies of hESCs may be accomplished with a KO serum-free culture system without the use of feeders by plating and growing the colonies on extracellular matrices (ECM) within a feeder-conditioned KO-DMEM medium supplemented with KOSR and FGF23. Furthermore, it has been suggested that feeder conditioning may be replaced by substituting the medium with high concentrations of FGF2 and noggin11. Alternatively, feeder conditioning was replaced by transforming growth factor β1 and human LIF (in addition to FGF2) and to growing the cells on human fibronectin7, or by serum-free media supplemented with soluble factors including FGF2, activin A, transforming growth factor β1 (TGFβ1), pipecolic acid, GABA, LiCL and culturing the cells on ECM components (16). In another recent publication, undifferentiated propagation of hESC colonies, in the absence of feeders, was reported with a chemically defined medium without serum replacer, supplemented with activin or nodal plus FGF212. In general, a key limitation of hESC culture systems is that they do not allow the propagation of pure populations of undifferentiated stem cells and their use typically involves some level of background differentiation. The stem cells most commonly follow a default pathway of differentiation into an epithelial cell type that grows either as a monolayer of flat squamous cells or form cystic structures. Most probably, this form of differentiation represents differentiation of hESC into extraembryonic endoderm8.
In these adherent culture systems of colonies, the hESCs are most commonly passaged (mechanically and/or by using enzymatic digestion) and re-plated as clusters, on a small scale. These culture systems are labor-intensive, highly variable, may contain undefined factors, and do not provide steady-state operating conditions. Most importantly, they do not typically allow for large scale production of standardized homogenous undifferentiated hESCs needed for the aforementioned uses.
Suspension culture bioreactors offer several advantages over the conventional use of static monolayer cultures. These systems facilitate the large-scale expansion of the cells in a homogeneous culture environment, thus decreasing the risk of culture variability. They are also less labor-intensive to operate and offer the possibility of computer control and monitoring of the culture conditions. Although bioreactors have been used to expand neural stem cells(17), mouse ES cells(18) and differentiating hESCs within embryoid bodies (EBs)(20), only recently some progress has been made towards the development of protocols for the feeder-free expansion of undifferentiated hESCs in suspension systems13.
A major obstacle in developing systems for culturing hESCs in suspension bioreactors was recently overcome. Until recently it was only possible to grow undifferentiated hESCs as flat cultures. Culturing clusters of hESCs in suspension induces their differentiation within EBs and is most commonly used to induce differentiation. Surprisingly, and against the accepted concept that hESCs in suspension undergo differentiation, it was recently demonstrated that hESCs may be propagated, in the pluripotent, undifferentiated state, as clusters in suspension using Neurobasal™ medium as the basic medium of the culture system(13), which is supplemented with N2. Neurobasal™ medium is a basal medium especially formulated for growth of neuronal cells, and supplemented with either serum (e.g., FBS) or B27 or N2 serum replacement23.